A single BIR domain of XIAP sufficient for inhibiting caspases (original) (raw)
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XIAP inhibits caspase-3 and -7 using two binding sites: evolutionarily conserved mechanism of IAPs
The EMBO Journal, 2005
The X-linked inhibitor of apoptosis protein (XIAP) uses its second baculovirus IAP repeat domain (BIR2) to inhibit the apoptotic executioner caspase-3 and -7. Structural studies have demonstrated that it is not the BIR2 domain itself but a segment N-terminal to it that directly targets the activity of these caspases. These studies failed to demonstrate a role of the BIR2 domain in inhibition. We used site-directed mutagenesis of BIR2 and its linker to determine the mechanism of executioner caspase inhibition by XIAP. We show that the BIR2 domain contributes substantially to inhibition of executioner caspases. A surface groove on BIR2, which also binds to Smac/DIABLO, interacts with a neoepitope generated at the N-terminus of the caspase small subunit following activation. Therefore, BIR2 uses a two-site interaction mechanism to achieve high specificity and potency for inhibition. Moreover, for caspase-7, the precise location of the activating cleavage is critical for subsequent inhibition. Since apical caspases utilize this cleavage site differently, we predict that the origin of the death stimulus should dictate the efficiency of inhibition by XIAP.
Mechanism of XIAP-Mediated Inhibition of Caspase-9
Molecular Cell, 2003
Department of Molecular Biology the caspase activation cascades. For example, active caspase-9 cleaves and activates caspase-3 and cas-Lewis Thomas Laboratory Princeton University pase-7. Thus, the activation and inhibition of the initiator caspases constitute a central regulatory step in cellular Princeton, New Jersey 08544 2 Department of Biology physiology. The inhibitor of apoptosis (IAP) family of proteins sup-370 Lancaster Avenue Haverford College presses apoptosis by inhibiting the enzymatic activity of both the initiator and the effector caspases (Deveraux Haverford, Pennsylvania 19041 3 Kimmel Cancer Center and Reed, 1999; Salvesen and Duckett, 2002; Shi, 2002b). At least eight members of the mammalian IAPs 233 S. 10 th Street Thomas Jefferson University have been identified, including X-linked IAP (XIAP), c-IAP1, c-IAP2, and Livin/ML-IAP. Each IAP protein con-Philadelphia, Pennsylvania 19107 tains one to three copies of the 80 residue zinc binding baculoviral IAP repeat (BIR). The different BIR domains Summary and segments in the same IAP protein appear to exhibit distinct functions. For example, the third BIR domain The inhibitor of apoptosis (IAP) proteins potently in-(BIR3) of XIAP potently inhibits the activity of the prohibit the catalytic activity of caspases. While profound cessed caspase-9 whereas the linker region between insight into the inhibition of the effector caspases has BIR1 and BIR2 selectively targets the active caspase-3 been gained in recent years, the mechanism of how or -7 (Fesik and Shi, 2001). The IAP-mediated inhibition the initiator caspase-9 is regulated by IAPs remains of all caspases can be effectively removed by the mitoenigmatic. This paper reports the crystal structure of chondrial protein Smac/DIABLO, which is released into caspase-9 in an inhibitory complex with the third bacuthe cytoplasm during apoptosis (Chai et al., 2000; Du loviral IAP repeat (BIR3) of XIAP at 2.4 Å resolution. et al., 2000; Verhagen et al., 2000). The proapoptotic The structure reveals that the BIR3 domain forms a activity of Smac/DIABLO depends on a 4 amino acid heterodimer with a caspase-9 monomer. Strikingly, IAP binding motif located at the N terminus of the mature the surface of caspase-9 that interacts with BIR3 also protein (Liu et al., 2000; Shi, 2002a; Wu et al., 2000).
Structural Basis of Caspase-7 Inhibition by XIAP
Cell, 2001
The inhibitor of apoptosis (IAP) proteins suppress cell death by inhibiting the catalytic activity of caspases. Here we present the crystal structure of caspase-7 in complex with a potent inhibitory fragment from XIAP at 2.45 A resolution. An 18-residue XIAP peptide binds the catalytic groove of caspase-7, making extensive contacts to the residues that are essential for its catalytic activity. Strikingly, despite a reversal of relative orientation, a subset of interactions between caspase-7 and XIAP closely resemble those between caspase-7 and its tetrapeptide inhibitor DEVD-CHO. Our biochemical and structural analyses reveal that the BIR domains are dispensable for the inhibition of caspase-3 and -7. This study provides a structural basis for the design of the next-generation caspase inhibitors.
Structural Basis of Caspase Inhibition by XIAP
Cell, 2001
The eliciting of caspase activities represents an evolutionarily conserved point of apoptotic cell death. These novel cysteine proteases specific for aspartic residues are the major players that disable homeostatic and repair processes, halt cell cycle progression, mediate
Journal of Cell Biology, 2002
he X-linked mammalian inhibitor of apoptosis protein (XIAP) has been shown to bind several partners. These partners include caspase 3, caspase 9, DIABLO/ Smac, HtrA2/Omi, TAB1, the bone morphogenetic protein receptor, and a presumptive E2 ubiquitin-conjugating enzyme. In addition, we show here that XIAP can bind to itself. To determine which of these interactions are required for it to inhibit apoptosis, we generated point mutant XIAP proteins and correlated their ability to bind other proteins with their ability to inhibit apoptosis. Ѩ RING point mutants T of XIAP were as competent as their full-length counterparts in inhibiting apoptosis, although impaired in their ability to oligomerize with full-length XIAP. Triple point mutants, unable to bind caspase 9, caspase 3, and DIABLO/HtrA2/ Omi, were completely ineffectual in inhibiting apoptosis. However, point mutants that had lost the ability to inhibit caspase 9 and caspase 3 but retained the ability to inhibit DIABLO were still able to inhibit apoptosis, demonstrating that IAP antagonism is required for apoptosis to proceed following UV irradiation. *Abbreviations used in this paper: IAP, inhibitor of apoptosis protein; BIR, baculoviral IAP repeat; TNF, tumor necrosis factor; TRAF, TNF receptor associated factors; XIAP, X-linked mammalian inhibitor of apoptosis protein.
2007
During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp 330 , and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp 330 removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp 330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp 315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp 315 , suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.
Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants
Biotechnology and Bioengineering, 2002
Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors. Such cell death may occur from necrosis but is more commonly associated with apoptosis. During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell. XIAP is the most potent caspase inhibitor encoded in the mammalian genome. The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells. CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation. In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin. Two deletion mutants of XIAP were created; one containing three tandem baculovirus iaprepeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs. Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment. Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases. Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments. With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns. While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus. In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol. Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 704–716, 2002; DOI 10.1002/bit.10154
Interaction with XIAP prevents full caspase‐3/‐7 activation in proliferating human T lymphocytes
European Journal of Immunology, 2008
Caspases are essential mediators of cytokine release and apoptosis. Additionally, caspase activity is required for the proliferation of naive T lymphocytes. It remained unclear how proliferating cells are able to cope with the pro-apoptotic activity especially of effector caspases-3 and-7. Possible reasons might include limited subcellular localization of active caspases or inhibition by endogenous caspase inhibitors. Here, we compared the activation of various caspases in proliferating human T cells with that in apoptotic cells. We show that cleaved caspases-3/-7 appear to be widely distributed in apoptotic cells while they are largely confined to the cytoplasm in proliferating cells. Additionally, in proliferating T cells caspase-3 remains incompletely cleaved, while in apoptotic cells fully mature caspase-3 is generated. We provide evidence that during T cell proliferation the intracellular caspase inhibitor X-linked inhibitor-of-apoptosis protein (XIAP) interacts with caspases-3/-7, thereby blocking their full activation, substrate cleavage, and cell death. The lack of substrate cleavage might also lead to the observed limited subcellular distribution of caspases-3/-7. After induction of apoptosis, second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with low isoelectric point (Smac/ DIABLO) is released from mitochondria, resulting in the abrogation of the inhibitory effect of XIAP, full activation of caspases-3/-7, and apoptosis.
The structure of XIAP BIR2: understanding the selectivity of the BIR domains
Acta Crystallographica Section D Biological Crystallography, 2013
XIAP, a member of the inhibitor of apoptosis family of proteins, is a critical regulator of apoptosis. Inhibition of the BIR domain-caspase interaction is a promising approach towards treating cancer. Previous work has been directed towards inhibiting the BIR3-caspase-9 interaction, which blocks the intrinsic apoptotic pathway; selectively inhibiting the BIR2-caspase-3 interaction would also block the extrinsic pathway. The BIR2 domain of XIAP has successfully been crystallized; peptides and small-molecule inhibitors can be soaked into these crystals, which diffract to high resolution. Here, the BIR2 apo crystal structure and the structures of five BIR2-tetrapeptide complexes are described. The structural flexibility observed on comparing these structures, along with a comparison with XIAP BIR3, affords an understanding of the structural elements that drive selectivity between BIR2 and BIR3 and which can be used to design BIR2-selective inhibitors.